Antibodies have found great use in the area of diagnostics and for assaying for the presence of antigenic materials in samples such as those derived from biological fluids and for research purposes. A drawback to such successes, however, has been the occurrence of unwanted side reactions in addition to the intended specific reactions of the antibody, which lead to the localization of the antigens under investigation by the specific antibodies used in the assays and the like.
Such unwanted side reactions can include unintended specific reactions, which are frequently considered as cross-reactivity, and which, under certain circumstances, may provide useful information regarding the variations of the structures of the materials under study. Here, the specific binding agent (e.g., the specific antibody used in the assay) recognizes epitopes in molecules other than the antigen under investigation. In addition, there is the often more troublesome unintended non-specific reactions governed by general physico-chemical properties, such as hydrophobic and electrostatic interactions between the antigen and the antibody. Methods to prevent cross-reactivity and non-specific reactions are essential to achieve the lowest background and so to optimize assay performance.
Fragments of immunoglobulins lacking the Fc region (e.g. Fab fragments) have been produced for diagnostic immunoassays for a variety of reasons. The most common reason is to eliminate interference from rheumatoid factor or other heterophilic antibody activity (cross linking), which most often occurs at the Fc region of the antibody. The Fc portion of IgG is also hydrophobic, which has high non-specific binding potential. None of the antigen binding domain of the immunoglobulin resides in the Fc portion; consequently, its removal has little or no effect on antigen binding affinity. One drawback of this approach is that preparation of purified Fab fragments is costly and time-consuming.
Protein A consists of a single polypeptide chain with little or no carbohydrate. The molecule is relatively heat stable and retains its native conformation even after exposure to denaturing reagents. The present invention solves the aforementioned problems by taking advantage of the discovery that Protein A is able to bind specifically to the Fc region of immunoglobulin molecules, which is distant from the antigen binding sites of such immunoglobulins. This invention discloses for the first time an immunoglobulin blocked with Protein A as well as processes using such a blocked structure in the immunological assay of selected analytes. Thus, the present invention relies on using protein A to block the Fc region in such a way as to suppress both cross-reactivity and the non-specific binding associated with this region, and to eliminate the need to generate Fab or other active fragments.
The present invention finds use in immunoassays carried out on microarrays of immobilized antibodies. Measurement of multiple antigens on these microarrays involves use of complex mixtures of antibodies for detection steps in the immunoassay. The use of such mixtures greatly increases the likelihood of non-specific signals, which can significantly reduce the sensitivity and dynamic range of the assay. We have shown that Protein A reduces the non-specific signals associated with the use of complex mixtures of antibodies.
In the past, a number of different molecular weight protein block such as BSA, gelatin, casein, non-fat dry milk, skim milk, normal serum, etc. have been used to prevent non-specific binding.
Protein A not only functions as a normal protein for blocking, but also has a number of advantages over the normal protein block:
1. By specifically binding the Fc portion of antibodies (which causes unintended specific binding) Protein A suppresses cross-reactivity without affecting antigen-antibody binding sites.
2. By specifically binding the Fc portion of antibodies, protein A changes the Fc region's physico-chemical properties (such as hydrophobicity) thereby reducing non-specific binding without affecting antigen-antibody binding sites.
3. With protein A blocking, the Signal/Noise ratio in immunoassays is significantly improved.